Accurate analyses of microbiota composition of low-density communities (103–104 bacteria/sample) can be challenging. Background DNA from chemicals and consumables, extraction biases as well as differences in PCR efficiency can significantly interfere with microbiota assessment. This study was aiming to establish protocols for accurate microbiota analysis at low microbial density.

Lower template concentrations significantly impacted microbiota profiling results. With higher dilutions, low abundant species were overrepresented. In samples of <105 bacteria per ml, e.g. DNA <1 pg/µl, microbiota profiling deviated from the original sample and other dilutions showing a significant increase in the taxa Proteobacteria and decrease in Bacteroidetes. In similar low density samples, DNA extraction method determined if DNA levels were below or above 1 pg/µl and, together with lysis preferences per method, had profound impact on microbiota analyses in both relative abundance as well as representation of species.

Conclusion

This study aimed to interpret microbiota analyses of low-density communities. Bacterial density seemed to interfere with microbiota analyses at < than 106 bacteria per ml or DNA <1 pg/µl. We therefore recommend this threshold for working with low density materials. This study underlines that bias reduction is crucial for adequate profiling of especially low-density bacterial communities.